Download Staining Reactions of Micro-Organisms

Staining Reactions of MicroOrganisms
Staining Reactions of Microorganisms
• All staining techniques begin with preparing the sample. This involves one
of two methods:
Method 1:
1. Mark a target circle on a microscope slide with a China marking pencil
about the size of your thumbnail.
2. Flame a bacteriological loop from the loop to where it joins the handle.
You want to get each area red-hot.
3. Let it cool to room temperature WITHOUT touching anything.
4. Dip it into a sample of water and place the loop-ful of water in the target
circle.
5. Reflame the loop and let cool.
6. Carefully remove a single colony of bacteria (certainly no more than the
amount of pus you'd get out of a medium sized zit when it pops) from
your solid media sample and place it in the water in your target circle.
7. Mix the water and bacteria together inside the target circle to make a thin
smear.
8. Let air dry.
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Method two:
1. Instead of using water and bacterial colonies, use a drop of liquid media
that has bacteria growing in it.
2. To remove the bacteria-containing sample, you remove the cap with the
pinky finger of your strong hand and pass the open neck of the tube
through the flame 2-4 times in your weak hand.
3. Dip your flamed loop into the broth with the other fingers of your strong
hand, remove the sample without touching the sides or neck of the tube
and reflame the neck of the tube.
4. Replace the cap on the tube and smear the sample inside your target
circle.
5. Flame your loop to re-sterilize it.
6. Allow the sample to air dry.
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• Regardless of the staining technique, all slides
are prepared as above.
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• With the exception of the capsule stain, the next step in the
majority of microbiological staining is to heat fix the slide.
• This process kills the bacteria and fixes them to the slide so
they won't wash off during staining or rinsing.
• To do this, you pass the slide through the flame of a Bunsen
burner 2-5 times or until it feels like baby's milk on your wrist
-- touch it carefully to your wrist so you don't get burned!
• Once you've heat fixed the slide, you may stain it as directed.
• Below (on the following slides) are the techniques for the
Simple stain, Gram stain, Acid-fast stain and Spore stain.
• All of these techniques work best in our little part of the world
-- techniques vary by location, altitude and quality of
chemicals.
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Simple Stain
• The first method is the Simple stain.
• This stain is very ... SIMPLE ... to perform, hence its
name.
• To your heat-fixed slide, add methylene blue to the
target circle for 1 minute.
• After the one-minute is past, rinse it with water and
blot it dry with bibulous paper.
• It's ready for viewing under the microscope on oilimmersion.
• Everything in this slide will appear blue.
• The only information you can get from this method is
the morphology of the microorganisms.
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For the Student: Gram Stain Summarized
• The primary dye here is crystal violet. The mordant (a chemical that makes the
dye stick better) is Gram's iodine. The counterstain (the stain that will stain
another color after a decolorization step is used) is safranin. There are many
techniques for this method. Indeed it is one of the simpler, faster, but most
under-utilized diagnostic stain method around. As long as the Gram's iodine is
left on the sample for as long as the crystal violet was, this technique is very
amenable to flexibility. Pour crystal violet onto the target circle and leave it
there for 15 seconds to 1 minute. Rinse it off with water. Pour Gram's iodine
on the target circle and leave it there for 15 seconds to 1 minute. Rinse it off
with water. The next two steps are critical, time-wise. Decolorize the stain
with acetone-alcohol for 3-5 minutes, then immediately do one of the
following: 1) rinse with water and then stain with safranin for 30 seconds or 2)
immediately stain with safranin for 30 seconds. Then rinse the safranin off
with water, blot dry with bibulous paper and the sample is ready for
examination on the microscope under oil-immersion. The information one
gets here is not only morphology, but also Gram reaction: purple is Gram
positive and pinkish-orange is Gram negative. This information may be utilized
in a sort of "choked shotgun" manner to prescribe more specific antibiotics for
bacterial infections than if one simply went by statistics and used a more
broad-spectrum antibiotic to "shotgun" the infection.
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• As mentioned above, there are two classes of bacteria by this
method: 1) Gram positive (purple because they retain the
crystal violet after decolorization) and 2) Gram negative
(pinkish-orange because they lose the crystal violet during
decolorization and take the safranin counterstain).
• Gram positive species vary in the tenacity with which they
retain the violet stain and some may be largely decolorized.
Additionally, older cultures give rise to false negative
reactions: it is usually safe to assume that organisms giving a
doubtful reaction are Gram positive.
• Furthermore, bacteria are stained evenly and deeply by basic
dyes. Basophilia is due to the high amount of RNA that is
distributed uniformly through the cytoplasm.
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There are differential qualities between the two
classes of Gram classified bacteria:
1. The composition of the cell walls is different: gram
positive bacteria are high in teichoic acid and
peptidoglycan and little to no lipopolysaccharide
(LPS); gram negative bacteria have no teichoic acid,
are low in peptidoglycan and are high in LPS.
2. Gram positive organisms have less synthetic ability
and so more complex nutritional requirements.
3. Gram positive bacteria produce primarily exotoxins
(there are exceptions); gram negative bacteria
produce only endotoxins.
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4. Gram positive bacteria are very resistant to
mechanical damage -- this explains why they
remain on our skin regardless of how well we wash
and scrub.
5. The two types of bacteria have different spectra of
susceptibility to chemotherapeutic agents,
disinfectants, dyes, simple chemicals and enzymes.
6. Gram positive bacteria tend to be more susceptible
to PCN; gram negative bacteria tend to be more
susceptible to TET (tetracycline).
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•
The value in this system is that there are only four
different "kinds" of bacteria: Gram positive rods or
cocci and Gram negative rods or cocci. In General:
1. All cocci of medical importance are gram positive
EXCEPT for the genera Neisseria, Branhamella (now
Moraxella) and Veillonella.
2. All rods of medical importance are Gram negative
EXCEPT for the genera Lactobacillus,
Mycobacterium, Corynebacterium, Bacillus and
Clostridium.
3. If the Gram stain is questionable it is reported as
gram positive.
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Endotoxins vs Exotoxins
•
Endotoxins are part of the cell
walls (LPS; Figure right) and
are released upon bacterial
death. They are released only
from gram negative bacteria.
They are very temperature
stable: at temperatures above
60 C, they are still toxic. They
have very low antigenicity, so
vaccines are uncommon. 10's
to 100's of micrograms cause
death. They raise body
temperature (cause fever)
increase
hemorrhage,
increase swelling in tissues
and induce vomiting and
diarrhea. Typical bacteria
include coliform bacteria. The
effects of LPS, systemically,
are summarized in Figure,
right.
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• Exotoxins are excreted by the living cell into its environment. These
toxins are excreted in high concentrations.
• They are produced by gram-positive bacteria (only rarely by gram
negative bacteria). Their composition is polypeptide and they are
unstable at temperatures above 60°C.
• They have very high antigenicity and are used in the formation of
antitoxins (non-toxic toxoids) by heat treatment, formalin or ether
treatments for immunizations.
• Less than microgram quantities cause death.
• It has been approximated that a 6-oz. bottle of Clostridium
botulinum exotoxin would be enough to decimate the world's
population.
• Exotoxins are located in the cytosol of the living cell. They interfere
with synapses, protein synthesis (translation), they increase
capillary permeability and increase water elimination.
• Typical bacteria include the spore formers, e.g., aerobic spore
formers (Bacillus) and the anaerobic spore formers (Clostridium).
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The table, below, summarizes exotoxins released from various
microbes and the disease caused by them:
Exotoxin
Example
Gram
reaction
Effected
structure
Disease
Neurotoxin
C.
botulinum
+
Neuromuscular
junction
Botulism
Neurotoxin
C. tetani
+
CNS
Tetanus
a toxin
C.
perfringens
+
General
Gas gangrene
Diphtheria
toxin
C.
diphtheriae
+
General
Diphtheria
Enterotoxin
S. aureus
+
Nerve cells
Food
poisoning
a toxin
S.
pyogenes
+
General
Pyogenic
infections
Hemolysins
Strep/Staph
+
Lyse RBC
Septicemia
Guinea pig
toxin
Y. pestis
-
General
Bubonic
plague
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ASIDE
• Endemic disease is when the disease is at a constant
in a constant geographical region with low morbidity
(diseased state) rate.
• Epidemic disease is when the disease has a morbidity
rate increased above "normal" with a high mortality
rate or will cause public harm.
• Pandemic disease is an over-grown epidemic, e.g.,
the geographical are has enlarged significantly, and
the number of people with the disease is increasing
at an alarming rate with increased mortality (death
rate) rate.
End of Aside
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Exotoxin Mechanisms of Action
• Exotoxins invade cells
by a mechanism
repeated throughout
nature: receptor binds
the exotoxin,
internalizes a part of
the exotoxin that exerts
its effects inside the
cell, right. In a nutshell,
the exotoxin binds to an
integral protein in the
cell membrane of the
host.
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Exotoxin Mechanisms of Action
• This binding causes a conformational
change in the integral protein which
permits the entry of a part of the
exotoxin (the portion that lacks cellbinding capability (the square in this
image), but that is responsible for the
toxic effects inside the cell; this
portion is enzymatically active) and
then releases another part of the
exotoxin (the portion that is non-toxic
and biologically inactive, but that is
required to cause the conformational
change in the integral protein
[receptor] to convert to a pore to
permit the biologically active portion
inside the cell to cause disease (the
triangle in this image)).
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Exotoxin Mechanisms of Action
• Examples of exotoxins
that work this way,
include cholera toxin
that increases cAMP
levels in the cell that
causes diarrhea and
botulinum toxin that
lowers Ach levels in the
NMJ and causes flaccid
paralysis.
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• Intracellularly, the exotoxin, e.g., Diphtheria toxin, exerts its
effects following internalization either by Receptor Mediated
Endocytosis or via internalization of the exotoxin once it is
detected by a clathrin-coated pit (similar to how LDL is taken
up in the liver), below.
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• Once the pit has internalized, the clathrin is recycled to the
cell surface and the endosome differentiates to a pH of about
7. Differentiation to a pH of about 5 continues at which point
the now acidic endosome differentiates to a CURL (review
A&P I).
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• The catalytically inactive portion of the exotoxin is released
from the cell. The catalytically active portion undergoes
processing, then interferes with translation. This interference
inhibits translation and causes cell death.
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Pore-Forming Toxins
• The first pore-forming exotoxin
causes electrolyte imbalance in
cells attacked by the bacterium.
• The bacterium first secretes the
"pre-pore" unassembled.
• When it "recognizes" the host cell
membrane, the pre-pore
undergoes a conformational
change and inserts itself into the
membrane -- much like a spigot
into a maple tree to collect maple
sap.
• When it is completely inserted
inside the membrane, the prepore undergoes another
conformational change that
opens the pore.
• This allows ions and other
particles to both exit and enter
the cell, causing its death.
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• A blow-up of the prepore. Note the presence
of linker amino acid (AA)
sequences that hold the
pre-pore "pieces"
together. These
sequences have no
classical secondary
structure. The function of
the N-terminal and Cterminal sequences is
that of a signal sequence.
The signal sequence is
the portion of the protein
that "identifies" the
membrane into which the
pore will be inserted.
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• The pore portions,
represented by the
wedges, have a
functional amino acid
sequence that gives the
pore its function. All 3 of
the proteins are
complimentary, so that
attractive forces will
"fuse" them together in
the membrane, making
the wedge that will,
upon complete
insertion, change its
shape to make the pore.
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• Figure, right, illustrates
perhaps the scariest of the
exotoxin-derived pores: the
bacterial escape pore
(BEP).
• Remember that bacteria
are not normal inside our
tissues or our blood.
• When they are detected in
our tissues or blood,
phagocytes are mobilized
to inactivate these bacteria
by phagocytosis.
• Once the bacterium has
been internalized, it is
temporarily stored in a
phagosome (an eating
pocket).
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• In some instances, the
phagosome will
differentiate into a
vacuole. It is while in
the vacuole that the
bacterium releases its
BEP. Much like the pore
that causes cellular
electrolyte imbalances,
the pore is released in
a "pre" form, it
arranges itself
complimentarily and
inserts itself into the
vacuolar membrane.
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• Once it is inserted,
it changes shape,
again, and the
bacterium escapes
the vacuole and
phagocytosis,
infecting the cell
and expediting its
death.
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• Organisms of the genus
Mycobacterium don't stain well
by Gram's method, but take up
hot carbol fuchsin and hold it so
well that they resist
decolorization with strong
mineral acids (nitric, hydrochloric
or sulfuric acid with ethyl
alcohol).
• Point of caution: ethyl alcohol
(EtOH) is lipophilic.
• Each of the three acids is caustic
to skin.
• Since they are mixed with EtOH,
the potential burn that this can
cause will be worse than just
getting burned by the acids alone.
• That's because the EtOH "drags"
the acids deeper into the skin
faster than by themselves.
Acid-Fast Stain
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Acid-Fast Stain
• Place your heat-fixed slide
on the top of a beaker with
boiling water in it. Cover the
target circle with carbol
fuchsin and keep it moist as
it heats. Continue boiling
the water beneath the slide
for 10 minutes. After the 10
minutes have passed,
remove the slide and allow
it to cool to room
temperature. Rinse it, then,
with acid-alcohol for about
30 seconds or until no more
fuchsia color comes from
the target circle.
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Acid-Fast Stain
• Counter-stain with
methylene blue for one
minute, then rinse with
water and blot dry with
bibulous paper and the
sample is ready for
examination under the
microscope on oil
immersion.
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• What you'll see, here, are a
blue background and
Mycobacterium
tuberculosis and other acidfast bacteria (AFB) show up
as bright red/fuchsia rods
that show up against this
blue background, i.e., the
acid alcohol does not
remove the fuchsia from
these microorganisms. This
method may be modified by
using a fluorescent dye in
place of the carbol fuchsin.
This may then be
illuminated with UV light.
This method is much
simpler to use for the
detection of AFB.
Acid-Fast Stain
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Spore Stain
•
The last staining technique to be discussed is the spore stain. This stain is for the
observation of free spores in the media or endospores -- spores inside the
bacterium -- which is indicative of infections that are of great seriousness. Place
your heat-fixed slide on the top of a beaker of boiling water and cover the target
circle with malachite green. Keep it moist during the 10 minute boiling period. After
10 minutes, remove the slide from the top of the beaker and allow it to cool to
room temperature. Rinse the slide until no more green comes off the target circle,
then counterstain with basic fuchsin for 1 minute. Rinse it off with water, blot dry
with bibulous paper and it's ready for viewing under the microscope on oil
immersion. The resulting spores and endospores are stained green and the
sporangia/bacteria are stained fuchsia.
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Bacterial Movements
1. True Motility: progression of bacterium
relative to other organisms -- due to flagella.
2. Brownian Movement: jerky movement due
to molecular bombardment.
3. Streaming Movements: in one direction due
to currents in the fluid.
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• The chief motile bacteria include most
varieties of E. coli, nearly all species of
Salmonella, Proteus, Pseudomonas, Vibrio and
Campylobacter, and some species of Bacillus
and Clostridium.
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• The chief non-motile bacteria include all
species of Shigella, Brucella, Haemophilus and
Mycobacterium, nearly all species of
Corynebacterium, and all cocci of medical
importance. Organisms with obvious capsules
do not produce flagella and are non-motile,
e.g., K. pneumoniae, C. perfringens and B.
anthracis.
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• Motile bacteria possess chemosensors and show
positive and negative chemotaxis, i.e., they move
towards some chemicals (mostly sugars and amino
acids; positive chemotaxis) and are repelled by
others (harmful substances and bacterial excretory
substances; negative chemotaxis).
• In terms of their pathogenicity and power of invading
the tissues, motile bacteria show no advantage over
those that do not possess flagella.
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